Transmembrane adapters: structure, biochemistry and biology

Seminars in Immunology 16 (2004) 367–377

Transmembrane adapters: structure, biochemistry and biology Stefanie Kliche, Jonathan A. Lindquist, Burkhart Schraven∗ Institute of Immunology, Otto-von Guericke-University, Leipziger Strasse 44, 39120 Magdeburg, Germany

Abstract Transmembrane adapter proteins (TRAPs) represent a relatively new and unique group of signalling molecules in hematopoetic cells. They differ from other signalling proteins as they lack any enzymatic or transcriptional activity, instead they possesses multiple tyrosine-based signalling motifs (TBSMs). Triggering of immunoreceptors induces tyrosine phosphorylation of these motifs by members of the Src-, Syk- or Tec-family of protein tyrosine kinases thus enabling the TRAPs to recruit cytosolic adapter and/or effector molecules via their SH2-domains into close proximity to the immunoreceptors, a position from which they can coordinate and modulate signal transduction pathways important for lymphocyte function. © 2004 Elsevier Ltd. All rights reserved. Keywords: Adapter proteins; TRAP; CAP; TCR; BCR; T-cell and B-cell

1. Transmembrane adapter proteins During the last years much has been learned about the molecular signalling events leading to T- and B-cell activation. A major focus of research has been the elucidation of intracellular signalling events following ligation of immunoreceptors (e.g. the T-cell receptor (TCR), or the B-cell receptor (BCR)). This had led to the identification of a group of proteins, which have collectively been termed adapter proteins. Adapter proteins lack either enzymatic or transcriptional activities, but are capable of mediating noncovalent protein–protein interactions with other signal transducing molecules via tyrosine-based signalling motifs (TBSMs) Abbreviations: BCR, B-cell receptor; BM, bone marrow; CAPs, cytoplasmatic adapter proteins; ITIMs, immunoreceptor tyrosine-based inhibition motifs; ITAMs, immunoreceptor tyrosine-based activation motifs; LAT, linker of activation of T-cells; LAX, linker for activation of X; LIME, Lck interacting membrane protein; NTAL/LAB, non-T-cell activation linker/linker for activation of B-cells; PAG/Cbp, phosphoprotein associated with glycosphingolipid-enriched microdomains/Csk binding protein; PLC, phospholipase C; PTKs, protein tyrosine kinases; SIT, SHP2 interacting transmembrane adapter protein; TBSMs, tyrosine-based signalling motifs; TCR, T-cell receptor; TRAPs, transmembrane adapter proteins; TRIM, T-cell receptor interacting molecule; ZAP-70, zeta-associated protein of 70 kDa ∗ Corresponding author. Tel.: +49 391 67 15800; fax: +49 391 67 15852. E-mail address: [email protected] (B. Schraven). 1044-5323/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.smim.2004.08.017

or modular protein–protein interaction domains (e.g. SH2-, SH3-, PH-, WW-, PTB- and PDZ-domains). The major function of adapter proteins is thus to facilitate the formation of multicomponent signalling complexes that allow the externally applied signal to be transduced from the cell surface into the intracellular environment. Adapter proteins are divided into two groups: transmembrane adapter proteins (TRAPs) and cytoplasmatic adapter proteins (CAPs) (for review see [1–7]). This review will exclusively focus on the role of transmembrane adapter proteins during the activation and differentiation of hematopoetic cells. The common features of all transmembrane adapter proteins known so far are the presence of a short extracellular domain, a single membrane-spanning region, and multiple TBSMs within the cytoplasmatic tail (Fig. 1 and Table 1). A subgroup of TRAPs possesses a conserved CxxC motif at the transmembrane cytosolic interface, which becomes palmitoylated. This motif is responsible for the localisation of certain TRAPs into glycosphingolipid-enriched microdomains (GEMs also termed lipid rafts) (Fig. 1). Immunoreceptor-induced cellular activation of protein tyrosine kinases (PTKs) of the Src-, Syk- and Tec-family results in rapid phosphorylation of the TRAPs which then mediate interactions with the SH2-domains of intracellular signalling molecules. By binding to the phosphorylated TRAPs the latter proteins are translocated to the inner side of the plasma

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Fig. 1. Schematic structure of the transmembrane adapter proteins (TRAPs) described in this review and their biochemical properties. Lipid rafts and nonassociated TRAPs are depicted and tyrosine-based signaling motifs (TBSMs) for each TRAPs are indicated. Amino acids (aa) are presented in single letter code.

membrane, where they come into close proximity with the activated immunoreceptors and/or their physiologic substrates. Seven transmembrane adapter proteins have been identified so far: Linker of Activation of T-cells (LAT), Non-Tcell Activation Linker/Linker for Activation of B-cells (NTAL/LAB), Lck Interacting Membrane protein (LIME), Linker for Activation of X (LAX), SHP2 Interacting Transmembrane adapter protein (SIT), T-cell Receptor Interacting Molecule (TRIM), and Phosphoprotein Associated with Glycosphingolipid-enriched microdomains/Csk binding protein (PAG/Cbp) (Fig. 1) [8–17]. In this review, we summarize our current knowledge of the structure, biochemistry and biology for these TRAPs.

2. Transmembrane adapter proteins associated with lipid rafts 2.1. LAT LAT is presently the best-characterized TRAP (for detailed reviews see [5,18,19]). LAT expression is detected

in the thymus, peripheral blood lymphocytes and spleen. In addition to thymic and peripheral T-cells, LAT is also found in mast cells, natural killer cells (NK-cells), megakaryotes, platelets and bone marrow (BM)-derived pre-B-cells [20–23]. LAT possesses a conserved CxxC motif which becomes palmitoylated and is responsible for the translocation into lipid rafts as well as for its proper function [24,25] (Fig. 1). The cytosolic tail of LAT contains nine TBSMs (Fig. 1) which become phosphorylated by zeta-associated protein of 70 kDa (ZAP-70) or Syk kinase (in non-T-cells) after external ligation of immunoreceptors. The sites of ZAP-70 phosphorylation have been mapped to Y127 , Y132 , Y171 , Y191 , and Y226 , respectively [16,26,27]. Cross-linking of CD28 also induces tyrosine phosphorylation of LAT, but apparently independently of ZAP-70 and Syk. It has been suggested that Itk (a member of the Tec-PTKs) could phosphorylate LAT upon CD28 stimulation [28,29]. Phosphorylation of LAT recruits several signalling molecules to the plasma membrane. Grb2, Gads, Grap, phospholipase C␥1 (PLC␥1), the p85 subunit of the PI3-kinase, 3BP2, Vav, and Shb directly bind via their SH2-domains to LAT. The binding sites for Grb2, Gads, PLC␥1, p85 sub-

Other binding partners: TCR␥, CD3␥/␦/␧

Y373 ENV → Grb2/Gads

Y268 VNM → PI3K Y294 ENV → Grb2/Gads

Y71 LRV → ? Y93 DIL → ? Y150 AVG → ? Y155 DNA → ? Y193 VNV → Grb2/Gads GNL→Grb2 GNL→? Y127 TSL→? Y79 EQM→PI3K VKY148 SEVa → SHP2 Y110 ASL → ? Y169 ASV → ? Y188 ANS → Grb2

Other binding partners: PI3K, SHP2, Grb2 ITIMS.

2.2. LAT: the master switch for T-cell development and T-cell activation

a Represent

Y387 EAI → ? Y233 VNG → Grb2 Y191 VNV → Grb2/Gads/Vav Y417 ESI → ? Y226 ENL → Grb2/Gads/Vav Other binding partners: Grap, 3BP2, and Shb Other binding partners: Fyn and EBP50

LAY235 QTL → Lck Y254 ESI → Lck (according to Hur et al.) Y136 ENV → Grb2 Y193 QNS → Grb2 Y341 TSI→? Y358 ATV→?

SNV → ? Y167 ARV → Csk VLY200 SRVa → Csk LAY235 QTLa → ? Y254 ESI → Lck, Fyn (according to Brdickova et al.) QQR → ? Y58 SLV → ? Y95 QNF → GrbZ Y110 IDP → ? Y118 YNW → Grb2 EEV→? EVL→? ETV→? ASV→? SSV→Csk Y163 Y180 Y227 Y317

DST→? Y45 PRQ→? Y64 PPV→? Y110 ENE → Grb2 Y127 HNP → Grb2

Y132 LVV → PLC␥ Y171 VNV → Grb2/Gads/PI3K

TRIM SIT

Y63 Y40

Y145

LIME NTAL/LAB PAG

Y105 Y36

LAT

Table 1 Tyrosine-based signalling motifs (TBSMs) and their (potential) binding partners for seven transmembrane adapter proteins (TRAPs)

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unit of the PI3-kinase and Vav have been identified (Table 1) [27,30–33]. The recruitment of these signalling molecules to LAT leads to an indirect binding of other important signalling proteins to couple TCR engagement to multiple intracellular signalling pathways, thereby regulating adhesion, cytoskeleton reorganization, Ca2+ influx, proliferation or AP-1-, NF-AT-, and NF-␬B-induced cytokine transcription (Fig. 2) (for detailed reviews see [1–4,19]). According to the currently accepted model, binding of SLP-76 to LAT via the small adapter protein Gads induces phosphorylation of SLP-76 by ZAP-70 to provide the binding site for the SH2-domain of Itk. Simultaneously, phosphorylated LAT binds PLC␥1 and the enzyme then becomes activated through dual phosphorylation by ZAP-70 and Itk. The activation of PLC␥1 leads to the generation of two second messengers, diacylglycerol (DAG) and inositol trisphosphate (IP3 ). While IP3 mediates Ca2+ flux, DAG activates the nucleotide exchange factor RasGRP, an activator of Ras. Next to the LAT-PLC␥1-DAG-RasGRP-Ras pathway, phosphorylated LAT binds Grb2 to recruit Sos for the activation of Ras. It thus seems that TCR-initiated signalling regulates Ras activation through two independent pathways, the LAT-PLC␥1-DAG-RasGRP Ras- and the LAT-Grb2-Sos-Ras-pathway (Fig. 2). However, binding of SLP-76 via Gads to phosphorylated LAT exerts additional activities during T-cell activation. Phosphorylated SLP-76 interacts with the cytosolic adapter protein Nck and with the nucleotide exchange factor Vav. This complex may be capable of activating the GTPase Rac1, thereby regulating the reorganisation of the cytoskeleton [34,35]. Finally, phosphorylated SLP-76 interacts with the adhesion and degranulation promoting adapter protein (ADAP) which is believed to alter the function of intergrins after TCRengagement [36–38]. At present it is unclear whether the SLP-76 associated portion of ADAP is involved in TCRmediated activation of integrins to promote adhesion or whether ADAP acts independently of its interaction with SLP-76. Further studies are required to answer this question.

Y90

LAX

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The generation and characterisation of LAT-deficient mice revealed the importance of LAT for T-cell development. LAT−/− thymocytes are blocked at the CD25+ CD44− (DN3) stage, where signalling through the pre-TCR drives the differentiation of DN to DP cells. As a consequence, these mice lack mature SP T-cells in the periphery [39]. The phenotype of knock-in mice with a LAT mutant in which the four distal tyrosine residues (Y136 /Y175 /Y195 /Y235 ) were mutated to phenylalanine (LATY4F ) is similar to the LAT−/− mice [40]. The phenotype of this mutant appears to be due to the inability of the LATY4F molecule to recruit PLC␥1, Gads/SLP-76 or Grb2/Sos to the plasma membrane. Thus, it seems as if the

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Fig. 2. Schematic illustration of the signalling events regulated through LAT in T-cells. Phosphorylation of LAT leads to the recruitment of the adapter proteins Grb2, Gads and PLC␥1. Gads is constitutively associated with SLP-76. Tyrosine phosphorylation of SLP-76 leads to association with Nck and Vav, a exchange factor for the GTPase Rac1. These events seem to be critical in the regulation of actin polymeriszation and cap formation of T-cells. Phosphorylated SLP-76 is also associated with ADAP, a CAP involved in the regulation of TCR-induced adhesion. Recruitment of Grb2 associated Sos to the LAT complex potentially gives rise to the activation of the Ras-Raf-ERK1/2 pathway, to enhance transcriptional activity of AP-1. Association of PLC␥1 and activation of this enzyme leads to the generation of two second messengers, diacylglycerol (DAG) and inositol trisphosphate (IP3 ). While IP3 mediates Ca2+ flux, DAG activates the nucleotide exchange factor RasGRP, an activator of Ras, to promote ERK1/2 activation. All pathways are closely connected to each other at several levels and contribute to the stimulation of transcriptional activity of NF-AT, AP-1 and NF-␬B resulting in IL-2 gene transcription.

four distal tyrosine residues of LAT are the most important for its proper function during T-cell development and T-cell activation. A recent report described the phenotype of knock-in mice expressing a LAT mutant, in which the three distal tyrosine residues Y175 /Y195 /Y235 were mutated to phenylalanine (LATY3F ) [41]. Mutation of these three tyrosine residues affects binding of the Grb2/Sos complex that links the TCR via Ras to the ERK1/2 activation as described above [32] as well as binding of the of the Gads/SLP-76 complex [32] that is required to activate PLC␥1 [42]. Thymic development in the LATY3F animals was found to be arrested at the DN3 stage as described in LAT−/− and in LATY4F mice [39,40]. However, in contrast to LAT−/− and the LATY4F mice, LATY3F thymi contain significant numbers of CD5low/ CD25+ ␥/␦Tcells that can also be found in the periphery. These ␥/␦T-cells possess an activated phenotype, produce large amounts of TH2 cytokines, but cannot be activated in vitro to proliferate or to express either CD25 or CD69 after TCR-stimulation. The LATY3F animals also lack dendritic epithelial cells (a thymus-dependent ␥/␦T-cell population) and ␥/␦ intraepithelial lymphocytes (IELs) which develop independently of the thymus [41]. These data suggest that LAT also controls the

development and activation of ␥/␦T-cells. Knock-in studies of single mutated distal tyrosine residue will be necessary to assess which of the three-tyrosine residues is/are responsible for controlling the development of this T-cell population. The importance of the interaction of LAT with PLC␥1 was analyzed using knock-in mice with a single Y136 substitution to phenylalanine (Y136F mice) [43,44]. Thymic development in these mice is also partially blocked at DN3. With age, a high number of CD4+ ␣/␤T-cells and a low number of CD5low/ CD25+ ␥/␦T-cells can be observed in the periphery of these mice. Interestingly, these T-cells also show an activated phenotype and secrete TH2 cytokines but do not flux calcium after TCR-stimulation. Later in life the LATY136F mice develop autoimmunity and the majority die from a massive infiltration of hematopoietic cells into tissues. At present the molecular basis for the LATY136F phenotype is not entirely clear and needs to be further elucidated. However, the occurrence of lymphoproliferative disorders in LATY3F and LATY136F animals might indicate that LAT possesses both positive and negative regulatory signalling functions which are necessary to control T-cell development (especially negative selection) and peripheral homeostasis of the immune system. With regard to the negative regulatory

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functions of LAT in peripheral T-cells, it is important to mention recent data which showed that LAT is capable of interacting with the adapter protein Gab2 and the tyrosine phosphatase SHP2 [45,46]. In the proposed model, the Grb2/Gab2/SHP2 complex inhibits TCR-mediated NF-AT activation and IL-2 production by competing with the Gads/SLP-76 complex for binding to LAT [45,46]. In line with this model is the observation that Gab2 expression becomes strongly upregulated in activated T-lymphocytes. Nevertheless, this model requires a more detailed analysis, especially because in other cellular systems the Gab2/SHP-2 interaction is believed to be involved in positive signalling rather than in signal inhibition (for a review see [47]). 2.3. LAT in non-T-cells Natural killer cells, mast cells and BM-derived pre-B-cells also express LAT and recent studies suggest that in these cells LAT plays a similar role in immunoreceptor-mediated activation. For example, stimulation of Fc␥RIII (CD16) on human natural killer cells leads to activation of Src- and Sykkinases, membrane recruitment of SLP-76 and activation of PLC␥1 [48,49]. LAT becomes phosphorylated upon ligation of Fc␥RIII and seems to participate in tyrosine phosphorylation of PLC␥1 [50]. A similar pathway has been found in mast cells following engagement of the high affinity receptor for IgE (Fc␧RI). Syk-mediated LAT phosphorylation after engagement of Fc␧RI leads (under involvement of SLP-76) to the translocation and activation of PLC␥1 and 2 [51–53]. This pathway results in a complex pattern of physiological responses, including degranulation and transcription of cytokines and chemokines [54,55]. Analysis of mast cells from LAT-deficient mice supports the view of a central role of LAT in Fc␧RI-mediated signalling. Loss of LAT in mast cells leads to a marked decrease in phosphorylation of SLP76 and impairs activation of the two isoforms of PLC␥. Accordingly, granule release and cytokine/chemokine production are impaired, but not fully blocked [53]. Retroviral reconstitution of LAT-deficient BM-derived mast cells (BMMCs) with the above described LATY4F mutant demonstrated that the four distal tyrosines are also critical for LAT-dependent signalling in mast cells. Single and double mutation of individual tyrosine residues showed their differential requirement for mast cell functions, with the primary PLC␥-binding site, Y136 , being the most important [55]. As described above, mast cell degranulation is not fully impaired in LAT-deficient mice, indicating that another TRAP might be involved in Fc␧RI-mediated signalling leading to granule release. One potential candidate would be the transmembrane adapter protein NTAL/LAB which we will describe below. A recent study has shown that LAT (and also the cytosolic adapter protein SLP-76) is involved in pre-B-cell development [23]. Su and Jumaa demonstrated that LAT and SLP-76 are expressed in BM-derived pre-B-cells where both adapter

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proteins constitutively interact with each other and become tyrosine phosphorylated after pre-BCR stimulation. Upon pre-BCR activation, the LAT/SLP-76 complex interacts with Ig␣ and with PLC␥2 to enhance calcium influx. Next to the Ig␣/SLP-65 pathway that has been described in mature Bcells [56], the LAT/SLP-76 pathway seems to represent an alternate pathway leading to the activation of PLC␥2 and Ca2+ mobilization in pre-B-cells. 2.4. Non-T-cell LAT? NTAL/LAB In contrast to pre-B-cells, mature B-lymphocytes do not express LAT. Therefore, one major question since LAT was discovered in 1998 was whether mature B-cells express a LAT-like TRAP that couples the BCR to intracellular signalling pathways. Two groups have recently reported the identification of a novel TRAP that could represent B-cell LAT. This protein was termed Non-T-cell Activation Linker (NTAL) by one group and Linker for Activation of B-lymphocytes (LAB) by the other [8,13]. NTAL/LAB (here referred as NTAL) is expressed in B-cells, NK-cells, macrophages and mast cells [8]. Like LAT, NTAL is localized to lipid rafts (due to the presence of a CxxC-motif that becomes palmitoylated) and possesses eight potential TBSMs motifs in the cytosolic domain (Fig. 1) which become phosphorylated by protein tyrosine kinases of the Syk-family (ZAP-70 or Syk) upon activation of the BCR or of Fc-receptors on macrophages or mast cells. Five TBSMs within the NTAL cytoplasmic domain represent potential binding sites for Grb2 (summarized in Table 1) and it has been shown that NTAL interacts with Grb2 upon immunoreceptor triggering [8]. In the A20 B-cell line tyrosine phosphorylation of NTAL occurs primarily on the three distal tyrosine residues and these residues were also found to be most important for the interaction of NTAL with Grb2 [57]. However, at present it is not clear whether NTAL represents B-cell LAT or not. Indeed, a number of observations argue against this possibility. For example, whereas NTAL possesses multiple Grb2 binding sites (similar to LAT) no consensus binding site for the SH2-domain of PLC␥1 or PLC␥2 is expressed. Also, coprecipitation experiments have revealed no evidence for an interaction between NTAL and Gads/SLP-76 or PLC␥, a hallmark of the LAT-induced signalling complex in T-lymphocytes (see Table 1 and Fig. 2). Finally, ectopic expression of NTAL in the LAT-deficient Jurkat T-cell line JCaM2.5 does not completely rescue the signalling defect in this cell line. Indeed, the experiments in JCaM2.5 cells have yielded conflicting results regarding the signalling function of NTAL. For example, Brdicka et al. showed that overexpression of NTAL in JCaM2.5 is capable of partially rescuing TCR-mediated ERK1/2 activation, but not calcium signalling [8]. In contrast, Koonpaew et al. showed that overexpression of NTAL in JCaM2.5 rescued Ca2+ influx but fails to induce ERK1/2 activation [57]. The reasons why so diver-

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gent results were obtained in the same cell line are unclear. It is however important to note that different modes of overexpressing NTAL/NTAL-mutants were applied by the two groups (retroviral transduction versus classical transfection) which might help to explain these results. Using retroviral reconstitution of LAT-deficient bone marrow cells with wild-type NTAL, Koonpaew et al. showed recently that NTAL is capable of restoring T-cell development in a LAT-deficient background. At least two of the three Grb2 binding sites must be present to allow NTAL to rescue thymic development in LAT-deficient mice [57]. However, unlike LAT reconstituted mice, peripheral T-cells reconstituted with NTAL constitutively express the activation marker CD69 but, on the other side, fail to produce IL-2 upon TCR stimulation. This suggests an altered homeostasis of the peripheral immune system in the NTAL reconstituted LAT−/− mice. In line with this view is the recent description of a LATdeficient mouse strain stably expressing NTAL under control of the CD2 promotor [58]. Similar to the data obtained by Brdicka et al. in JCaM2.5 cells, LATdeficient /NTAL+/+ T-cells show a rescue of ERK1/2 activation but fail to signal calcium after TCR-stimulation. Perhaps more importantly however is the finding of an increased secretion of TH2 cytokines and IgG1 as well as massive organomegaly along with a disruption of the lymphoid follicle architecture in these transgenic mice. Thus, the phenotype of the LATdeficient /NTAL+/+ transgenic mice is almost identical to that of the LATY136F knock-in mutant described above. This indicates that from a functional point of view NTAL resembles a LAT molecule carrying a defective PLC␥1 binding site. Given all these data it may be speculated that NTAL does not represent the long sought B-cell homologue of LAT, a question which certainly will by answered by the analysis of NTAL-deficient mice. Nevertheless, from the above hypothesis three questions emerge namely (i) does the BCR signal independently of a LAT like molecule or have we still not found the real “Bcell LAT”, (ii) if there is no B-cell LAT, does NTAL need help from another transmembrane adapters expressed in Blymphocytes (e.g. SIT and LAX, see below) to couple the BCR to downstream signalling pathways?, and (iii) what is the signalling function of NTAL in B-cells if it is not the “B-cell LAT”? With regard to the last question it is important to note that obviously some types of hematopoietic cells, for example mast cells and NK-cells, co-express LAT and NTAL. A recent report demonstrated that in mast cells both NTAL and LAT become phosphorylated after Fc␧RI triggering. Furthermore, RNAi-mediated inhibition of expression of either LAT or NTAL in these cells reduces Fc␧RI-mediated degranulation [59]. This supports the view that both molecules are required for optimal mast cell function. Interestingly, however, no additional decrease in degranulation was detected when the expression of NTAL and LAT was ablated concomitantly which might indicate that NTAL and LAT control the same Fc␧RI-mediated pathway leading to mast cell de-

granulation. Clearly, additional studies are required to solve this puzzle. However, as already mentioned above, it is known that Fc␧RI-mediated degranulation is not completely blocked in LAT-deficient mast cells which suggests that some aspects of mast cell function can occur in the absence of LAT. It is tempting to speculate that this redundant function is mediated by NTAL, a question that will be answered by the analysis of LAT/NTAL-double deficient mice. 2.5. PAG/Cbp, a negative regulator of T-cell activation Among the various TRAPs, PAG/Cbp (here referred as PAG) is the only one that is ubiquitously expressed. PAG possesses a short 16 aa extracellular domain, a single transmembrane region and a large cytoplasmatic domain (Fig. 1) [9,14]. A palmitoylation motif (CxxC) located next to the transmembrane region becomes palmitoylated in vivo and is believed to be responsible for the translocation of PAG into lipid rafts (Table 1). Furthermore, PAG contains two proline rich motifs (PxxP) and 10 tyrosine residues within the cytoplasmic domain. Nine of these tyrosine residues represent potential TBSMs (Fig. 1 and Table 1). Src-family kinases, but not members of the Syk-family have been shown to phosphorylate PAG. However, so far, only the C-terminal Src kinase (Csk), the Src-kinase Fyn, and erzin/radixin/moesin-binding phosphoprotein 50 (EBP50) have been reproducibly found to interact with PAG in vivo [9,14,60,61] (Table 1). Mutation of the individual TBSMs within the PAG cytoplasmic domain demonstrated that Csk binds to Y317 of human [9] or Y314 of rat PAG [14]. This interaction is mediate via the SH2 domain of Csk [62]. Since it had previously been shown that Csk mutants lacking an SH2 domain fail to translocate to the plasma membrane and to regulate T-cell receptor signalling [63], the identification of PAG as a constitutive binding site for Csk was an important finding, as this provided a potential mechanism by which Csk is targeted to its membrane-associated substrates (the Src kinases) to regulate their enzymatic activity [64]. In membranes prepared from resting T-cells, PAG is the most abundant phosphorylated protein. Moreover, several groups have shown that immediately upon T-cell activation PAG becomes dephosphorylated. This TCR-mediated decrease in PAG phosphorylation is accompanied by a concomitant reduction of PAG-associated Csk [9,60,62]. The model that has been deduced from these observations proposes that in resting T-cells the activity of the Src kinases is kept low due to phosphorylation of the negative regulatory tyrosine residues by Csk. After stimulation of the TCR, PAG is dephosphorylated by an unidentified phosphatase, thus releasing Csk. Recently, transgenic mice over-expressing wild-type or a PAG-mutant lacking the Csk-binding site were investigated for their ability to signal via the TCR. The results obtained with these animals further supported the above model and substantiated the role of PAG as a negative regulator of T-cell activation [65].

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The identification and characterization of the PAG phosphatase will be of major importance. A recent study suggested the PAG is primarily dephosphorylated by CD45, the major membrane-associated phosphatase expressed in T-cells and a counterplayer of Csk with regard to the regulation of Src kinase activity [65]. In contrast, using SHP2-deficient fibroblasts, Zhang et al. most recently suggested, that the cytosolic tyrosine phosphatase SHP2 is responsible for the dephosphorylation of PAG [66]. Thus, the question which phosphatase dephosphorylates PAG in vivo is still under debate. However, it needs to be considered that distinct phosphatases could dephosphorylate PAG in different cell types. Whether these is indeed the case requires further studies. In contrast to the regulated binding of Csk, mutation of any of the nine TBSMs of PAG has no effect on the interaction between PAG and the Src-kinase Fyn [9]. These data could suggest that Fyn binds via its SH3 domain to one of the proline-rich regions within the cytoplasmic tail of PAG. However, experimental evidence supporting this hypothesis is still missing. Nevertheless, the constitutively interaction between PAG and Fyn has been demonstrated to be essential for PAG phosphorylation [67]. The major question emerging from these findings is how Fyn-mediated PAGphosphorylation is regulated. Recently it has been shown that in resting T-cells, the majority of Fyn apparently localized within the lipid rafts, whereas the bulk of Lck is found within the detergent-soluble membrane fraction. Following TCR stimulation, Lck rapidly translocates into the rafts where it becomes activated. Importantly, this shuttling of Lck to the rafts seems to be required to activate the raft associated Fyn [68]. Thus, this model predicts that raft-associated Fyn acts downstream of Lck. The obvious question emerging from these data is how the constitutive high phosphorylation levels of PAG are maintained in resting T-cells. Possibly, in the absence of phosphatase activity the basal levels of Fyn-activity are sufficient to keep PAG in a phosphorylated state. Immediately after TCR-engagement, the above discussed PAGphosphatase could act on PAG before Lck shuttles to the rafts and thus release Csk from the membrane. This together with the translocation of Lck to the rafts would induce activation of Fyn, thus resulting in re-phosphorylation of PAG, re-recruitment of Csk to the membrane and inhibition of Srckinase activity. Clearly, additional studies, including detailed kinetics of PAG-dephosphorylation, Lck shuttling and Fyn activation after TCR-engagement are required to solve these puzzling questions. Solving these questions will certainly by complicated by the recent identification of LIME, another Csk-binding raft-associated TRAP that is believed to regulate Src-kinase activity (see below). An additional mechanism by which PAG may regulate TCR-mediated signalling is the interaction between the Cterminal VTRL motif of PAG with the PDZ domain of the adaptor protein EBP50, as shown by Brdickova et al. [61] and Itoh et al. [60]. Indeed, both studies showed that EBP50 not only interacts with PAG, but also with members of the ezrinradixin-moesin (ERM) family of proteins, thus providing a

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molecular link between the lipid rafts and the cytoskeleton. It has been proposed that in resting T-cells, the mobility of lipid rafts is kept low because they are anchored via EBP50 to the cytoskeleton. Upon TCR-activation the association with EBP50 is lost, allowing the lipid rafts to be aggregated within the immune synapse [60]. Indeed, PAG overexpression in Jurkat T-cells resulted in a reduced lipid raft aggregation and an inhibition of immune synapse formation [60]. However, the mechanism by which the PAG–EBP50 interaction is broken is still unclear.

2.6. LIME and the activation of Src-kinases Lck Interacting membrane protein (LIME) is the most recently identified transmembrane adapter protein [10,12]. Like LAT and NTAL, it possesses a CxxC motif required for raft targeting and a cytoplasmic domain containing five potential TBSMs, one of which appears to be an Immunoreceptor Tyrosine-based Inhibition Motif (ITIM) (Fig. 1 and Table 1). There are conflicting results between the two reports describing LIME regarding the expression pattern of this molecule. Whereas one group finds that LIME is expressed preferentially in T-cells (including DN, DP and SP thymocytes) and NK-cells, but not outside of the hematopoietic system (with exception of the liver) [10], the other group shows that LIME is expressed also in B-lymphocytes, lung, spleen, but not in thymocytes [12]. Further, one group observed expression in the Jurkat T-cell line, whereas the other group reported that Jurkat T-cells do not express this molecule. Conflicting data are also reported regarding the levels of expression in resting versus activated T-lymphocytes. Brdickova et al. reported that LIME expression is strong in resting T-cells and declines after prolonged TCR-stimulation using CD3 mAbs and IL-2, whereas Hur et al. suggest that LIME expression is low in resting T-cells, but becomes rapidly upregulated after CD3/CD28 co-crosslinking. So far, it is also not clear how LIME becomes phosphorylated. One group reports phosphorylation after CD3 stimulation whereas the other group suggest that LIME exclusively becomes phosphorylated after triggering of the CD4 and CD8 co-receptors and shows that CD3-stimulation rather induces a decrease in LIME-phosphorylation. The reasons for these conflicting results may be that both groups have used cells from different species (murine versus human peripheral T-cells and Jurkat T-cell lines) as well as different LIME-antibodies, and finally different modes of T-cell stimulation (CD3 + IL-2 versus CD3 + CD28). Further studies are certainly necessary to clarify these points. One of the two reports describing LIME showed that upon stimulation of the TCR, LIME associates with Lck, PI3 kinase, Grb2, Gads and SHP2 and activates ERK1/2 and JNK, but not p38. Moreover, overexpression of LIME in Jurkat Tcells induces activation of the interleukin-2 promotor. These data could suggest that LIME is a positive regulatory TRAP [12].

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Brdickova et al. reported that LIME simultaneously binds both Csk and Src kinases (Lck and Fyn) after CD4-triggering [10]. In line with this, an increased recruitment of Lck and Csk into the lipid rafts was found in Jurkat T-cells stably overexpressing LIME. In these cells, the LIME-associated fraction of Lck is strongly phosphorylated on its negative regulatory tyrosine residue, Y505 , but surprisingly, its kinase activity towards an endogenous substrate is not affected. Mutational analysis of individual tyrosine residues within the cytoplasmatic domain of LIME revealed that both Csk and the Src-kinases bind to LIME via their SH2-domains possibly providing a molecular explanation for these somewhat paradoxic data. Thus, whereas the negative regulatory tyrosine of the LIME-associated fraction of the Src kinase becomes phosphorylated by Csk, the SH2-domain is blocked because it interacts with another phosphorylated tyrosine residue of LIME. As a consequence, the closed conformation that downregulates Lck activity under normal circumstances cannot be formed and the kinase stays enzymatically active. Indeed, in Jurkat T-cells stably overexpressing LIME, many molecules were found to be constitutively tyrosine phosphorylated. This enhanced global tyrosine phosphorylation could be reverted using an inhibitor of Src-kinases, PP2, suggesting that it is dependent upon Src-kinase activity. In summary, the published data are compatible with the idea that LIME primarily serves as a positive regulator of T-cell activation. On the other hand, it has been known for some time that engagement of CD4 in the absence of a TCRmediated stimulus delivers an inhibitory signal into T-cells that impairs subsequent T-cell activation [69–72]. The molecular basis for CD4-mediated inhibition of T-cell activation is to a large extent unclear. Because of its ability to recruit Csk to the rafts after CD4-stimulation, LIME could represent a prime candidate for mediating the negative regulatory effect of the CD4 molecule. The analysis of CD4-signalling in LIME-deficient mice will help to clarify this issue. It is, however, important to note that PAG also serves an inhibitory function during T-cell activation by recruiting Csk to the lipid rafts (see above) [9]. Therefore, it might be necessary to generate LIME/PAG double-deficient mice to identify the inhibitory pathways (not only mediated via CD4) that are controlled by the two transmembrane adapter proteins.

3. Non-raft transmembrane adapter proteins 3.1. LAX The search for proteins possessing multiple Grb2 binding sites also yielded the identification of an additional adapter protein that was termed LAX [17]. The expression of LAX appears to be restricted to the hematopoietic system (T- and B-cells) although a detailed expression analysis has not been performed. Like other TRAPs, LAX does not possess the typical palmitoylation motif and therefore is not found in lipid rafts. In contrast to other TRAPs, the extracellular domain

of LAX is considerably longer (Fig. 1). The cytoplasmatic domain of LAX contains eight TBSMs, four of which represent potential binding sites for Grb2 (Fig. 1). LAX becomes phosphorylated by both Src and Syk family kinases upon TCR or BCR stimulation. The Y193 motif is identical to the Gads binding site of LAT and the Y268 motif in LAX represents a consensus binding site for the PI3-kinase p85 regulatory subunit (Table 1). Tyrosine phosphorylated LAX binds Grb2, Gads and PI3K after overexpression in Jurkat T-cells but only the interaction of Grb2 with endogenous LAX has been confirmed so far in vivo. Transient overexpression of LAX impairs TCR-mediated activation of the transcription factors NF-AT and AP-1 and selectively inhibits the activation of p38 [17]. The major question from the functional studies performed with LAX is how this TRAP impairs TCR-mediated signalling. The answer to this question will require a detailed structure function analysis of LAX and the generation and characterization of LAXdeficient mice. 3.2. SIT and TRIM, two non-raft brothers serving similar function? SIT is exclusively expressed in lymphoid organs, including thymus, spleen, and lymph nodes. SIT can also be detected in the Peyer’s patches and cells of the peritoneal cavity, but CD3+ splenic T-cells and plasma cell express the highest amount of SIT ([15] and Simeoni and Schraven, unpublished data). In contrast to the other known TRAPs, SIT is Nglycosylated on a single site within its extracellular domain. This glycosylation may represent a binding site for an unknown ligand. One other peculiar feature of SIT is that the molecule forms a disulfide-linked homodimer (Fig. 1). SIT possesses no CxxC motif (and thus is excluded from the rafts) and the cytoplasmatic tail contains five TBSMs which become phosphorylated by PTKs of the Src and Syk family after TCR triggering (Fig. 1 and Table 1) [15,73]. Two TBSMs of SIT represent consensus sites for Grb2, with the Y90 motif having been identified as the major Grb2 binding site. SIT also possesses one ITIM that has been shown to recruit the cytoplasmatic tyrosine phosphatase SHP2 when phosphorylated (Table 1) [15,73]. Overexpressing of wild-type SIT in Jurkat T-cells inhibits TCR-mediated induction of NF-AT and expression of a SIT Y148F (ITIM motif) mutant produces the same phenotype, even through SHP2 binding to SIT is abolished. Also, overexpressing of a SIT mutant in which only the YASV-motif remained intact had exactly the same inhibitory effect upon NF-AT activation. These data collectively suggested that SIT acts primarily as a negative regulator of TCR-mediated signalling and that the major functional motif of SIT is Y169 ASV. This hypothesis is supported by the recent analysis of SIT-deficient mice which show enhanced positive selection in particular TCR-transgenic models (L. Simeoni and B. Schraven, manuscript in preparation).

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As the name T-cell Receptor Interacting Molecule implies, TRIM was identified as a coprecipitating protein of the TCR. Northern and Western blot analysis revealed a strong expression of TRIM in the thymus at very early developmental stages (DN1–DN2 and DN2), in ␥/␦ T-lymphocytes and NK1.1+ ␣/␤ thymic T-cells. TRIM expression was also detected to a lesser extend in T-cells of the spleen, lymph nodes, and peripheral blood whereas TRIM was not detected in the bone marrow, fetal liver, in B-cells, or in monocytes. These data suggest that TRIM is specifically expressed in T-lymphocytes [11,74,75]. Like SIT, TRIM forms a disulfide-linked homodimer, and has no CxxC motif (Fig. 1 and Table 1). In response to TCR activation, TRIM becomes phosphorylated by members of the Src kinases family (Lck and Fyn), but not of the Syk family. The cytosolic domain of TRIM contains three TBSMs, one of which was identified to interact with the SH2 domain of the p85 regulatory subunit of the PI3-kinase after TCR engagement (Fig. 1 and Table 1) [11]. However, the functional relevance of this association needs to be further clarified. Coprecipitation experiments and confocal laser scanning studies originally indicated that TRIM specifically colocalizes with the TCR. The interaction of TRIM with the TCR occurs via the TCR␨ chain and to lesser extent via the CD3␥/␧ heterodimer. With domain swapping mutants it was shown that the extracellular, transmembrane and the cytosolic domains of TRIM are required to mediate this interaction to the TCR␨ chain [75]. After overexpression of wild-type TRIM in Jurkat T-cells, the expression level of the TCR is strongly increased, suggesting that TRIM stabilizes the TCR–CD3 complex on the cell surface [75]. Further biochemical studies suggested that TRIM upregulates the surface expression of the TCR by inhibiting its spontaneous internalization and not by enhancing the export of preassembled TCR complexes to the plasma membrane [75]. Overexpressing of TRIM not only enhances TCR expression, but also increases Ca2+ release after TCR stimulation. However, the increased Ca2+ signal of TRIM transfectants is not dependent on any of the three cytoplasmic TBSMs of TRIM [75]. These data suggested that TRIM might be involved in regulating TCR-mediated signalling by altering the expression level of the TCR on the surface of T-cells. However, TCR expression levels of both thymic and peripheral T-cells prepared from TRIM-deficient mice seem to be normal and neither selection processes nor TCRmediated signalling functions such as proliferation or calcium influx appear to be impaired (B. Schraven and U. K¨olsch, manuscript in preparation). Thus, the function of TRIM for T-cell activation and T-cell development remains elusive. One possible explanation for the lack of a phenotype in TRIM-deficient T-cells would be that another TRAP compensates for the loss of this adapter protein. A possible candidate would be SIT since both SIT and TRIM share many structural properties. Indeed, both molecules are excluded from the lipid rafts, both are expressed as disulfide linked homodimers and most notably, they share two TBSMs within their

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cytoplasmic domain, the YNGL- and the YASV-motif (which is YASL in TRIM (see Table 1)). Thus, it seems reasonable to propose that a TRIM/SIT-double deficient mouse will show a much clearer phenotype than the single knock-out animals.

4. Concluding remarks Due to space limitations we have focused on the major aspects regarding the signalling functions transmembrane adapter proteins. Clearly all available data suggest that LAT represents a master switch for T-cell activation and development whereas the other transmembrane adapter proteins seem to play less prominent roles in regulating these processes. However, given the fact that all TBSMs of the known TRAPs are highly conserved between mouse and man, it is reasonably to propose that the non-LAT transmembrane adapters also serve important (but less essential) roles within the immune system. It is tempting to speculate that these transmembrane adapters serve to finely tune the immune response, for example in particular immunological environments. Indeed, alterations of the phosphorylation status of these transmembrane adapter proteins, while not leading to a complete failure of the immune system, could well be involved in the onset or the maintenance of immunological disorders such as autoimmune diseases and/or chronic inflammation. The sophisticated analysis of single or combined knock-out, knock-in or transgenic mice as well as the use of animal disease models will help to clarify the functional role of the non-LAT transmembrane adapter proteins within the immune system. Another question certainly is whether all transmembrane adapter proteins expressed by hematopoietic cells have already been identified or whether we can expect the discovery of novel members of this exciting group of signalling molecules. Indeed, sophisticated search of public databases using appropriate algorithms could help to identify additional TRAPs or TRAP-like molecules. It is also important to keep in mind that some transmembrane adapter proteins (e.g. PAG and maybe also NTAL) are also expressed in non-hematopietic cells. While research on TRAPs has so far primarily focussed on their role in immune cell signalling it will be important in the future to assess their function also in other types of cells (e.g. cells expressing Srckinases). It is tempting to speculate here that TRAPs also play important signalling roles outside the hematopoietic system. If true, the molecular connection of “outside” with “inside” via TRAPs would turn out to be a general principle in cell biology.

Acknowledgements This work was supported in part by grants from the Deutsche Forschungsgemeinschaft (DFG) [Schraven 533/51 and 533/6-1] to B.S. We thank Dr. Martin Kliche for preparation of the figures.

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